+

WO2009117031A2 - Système et procédé pour le profilage de séquences nucléotidiques pour l’identification d’échantillons - Google Patents

Système et procédé pour le profilage de séquences nucléotidiques pour l’identification d’échantillons Download PDF

Info

Publication number
WO2009117031A2
WO2009117031A2 PCT/US2008/087434 US2008087434W WO2009117031A2 WO 2009117031 A2 WO2009117031 A2 WO 2009117031A2 US 2008087434 W US2008087434 W US 2008087434W WO 2009117031 A2 WO2009117031 A2 WO 2009117031A2
Authority
WO
WIPO (PCT)
Prior art keywords
fragments
nucleotide sequence
separation
template
detecting
Prior art date
Application number
PCT/US2008/087434
Other languages
English (en)
Other versions
WO2009117031A3 (fr
Inventor
Gary W. Procop
Wei Wei
Ho-Ming Pang
Original Assignee
Advanced Analytical Technologies, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Advanced Analytical Technologies, Inc. filed Critical Advanced Analytical Technologies, Inc.
Publication of WO2009117031A2 publication Critical patent/WO2009117031A2/fr
Publication of WO2009117031A3 publication Critical patent/WO2009117031A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the invention relates to the identification of samples by nucleotide sequence profiling.
  • biochemical and phenotypic techniques have been used for microorganism identification.
  • the conventional methods for microorganism identification are based on isolation, cultivation, colonial morphology, and subsequent biochemical testing (e.g., oxidase production and glucose fermentation). These methods, although effective, are slow and typically take more than 24 hours to generate enough data to achieve an accurate identification. In addition, they usually are not discriminatory enough to provide sub-species identification.
  • numerous different types of biochemical reactions must be maintained in a quality manner in the laboratory to correctly identify the sundry bacteria that may be encountered in a clinical specimen. Fortunately, these are now commercially- available in a variety of identification kits. Although commercial identification products most often represent a distinct advance over traditional tube testing, they are costly and errors in identification may occur.
  • genotypic techniques have emerged as the preferred methods for microorganism identification, because of the high-degree of accuracy provided, when DNA sequencing is used in conjunction with a sufficiently discriminating genetic target.
  • nucleic acid sequencing information for the identification of microorganisms, and this is considered by many the "gold standard" for taxonomic categorization.
  • the microorganism is cultured and the DNA is extracted from the microorganism and submitted for the polymerase chain reaction (PCR).
  • the PCR amplifies a section of genome that is characterized through the traditional DNA sequencing reaction (i.e. Sanger sequencing).
  • PCR amplification uses two primers that hybridize to opposite strands of a specific DNA section.
  • the primers are oriented so that the elongation reaction proceeds from 5' to 3' across the region between the two primers.
  • These primers are often designed to hybridize to conserved regions in the bacterial or fungal genome (i.e., they are broad-range primers). In addition, they are designed to flank a region or regions that contain sufficient sequence variation to afford organism characterization.
  • the PCR product is then submitted to the Sanger sequencing reaction (i.e., enzymatic chain elongation/ termination through the incorporation of di-deoxyribonucleotide triphosphate [ddNTP] incorporation).
  • the products of the Sanger reaction each have one of the four ddNTPs at the terminus of the DNA strand and the four ddNTP molecules are labeled with different fluorophores.
  • the products are then separated by electrophoresis and differentiated with laser-induced fluorescence detection.
  • Another method to label the Sanger reaction products for detection uses a fluorescently-labeled primer. In this case, only one ddNTP is used to terminate the reaction.
  • Four different reactions, each with a different ddNTP are used to determine the DNA sequence. The reactions products from the four reactions are pooled, then electrophoretic separation and detection is performed.
  • Maxam and Gilbert have developed a sequencing method based on chemical reactions. This reaction procedure determines the nucleotide sequence of a terminally labeled DNA molecule by random breaking at adenine (A), guanine (G), cytosine (C), or thymine (T) positions using specific chemical agents. This method initially modified the DNA with base-specific modification reactions then followed by the removal of the modified base from its sugar and cleaved the DNA strand at that sugar position. For each breakage, two fragments are generated from each strand of DNA. In order to perform a sequencing analysis, only one fragment has an associated label for later detection. The products of these four reactions are then separated with gel electrophoresis.
  • Sequencing methods either enzymatic chain elongation reactions or chemical reactions, allow for base position determination and, therefore, DNA sequence determination.
  • a sequencing instrument identifies the base from the specific emission wavelength corresponding to the four different ddNTPs or primers that are labeled with different fluorescent moieties that have a specific emission spectrum. Microorganisms can be identified though the DNA sequence, since the genomic information of particular loci is unique to individual species of microorganism.
  • primer labeled enzymatic chain elongation reactions and chemical sequencing methods four reactions for each dye labeled primers are required. After electrophoresis separation and fluorescence detection, four lanes of bands or peaks are observed as shown in Figure IA. A distinct color can represent each type of nucleotide.
  • the assign sequence could be CTTGATCTTCATGGTAGGCCTCCTGAATCCT.
  • Figure 2 represents a signal outcome from a DNA sequencing instrument wherein dye terminators are used for enzymatic chain elongation/termination reaction and all four reactions of primer labeled for enzymatic chain elongation reaction are pooled together. At any position, the highest color peak represents one of the nucleotides.
  • Embodiments of the invention provide for nucleotide sequence profiling for sample identification, obtained by a method comprising one or more of the steps of extracting a nucleotide sequence template from the sample, performing nucleotide sequence amplification on the template (e.g., via PCR) to create an amplified sample, performing less than four nucleotide-specific chemical cleavage reactions on the amplified template to obtain nucleotide sequence fragments, performing size-based separation on the sample fragments, detecting the sample fragments' separation, generating a profile based on the detection, and comparing the profile to a data base for sample identification.
  • a method is useful for efficiently identifying a sample such as a microorganism and/or gene.
  • Figure IA shows a prior art four lane electropherogram.
  • Figure IB shows a prior art single lane electropherogram.
  • Figure 2 shows a prior art DNA sequencing instrument output.
  • Figure 3 shows a theoretical electropherogram for Staphylococcus aureus in accordance with an embodiment of the invention.
  • Figure 4 shows a theoretical electropherogram for Staphylococcus epidermidis in accordance with an embodiment of the invention.
  • Figure 5 shows an electropherogram in accordance with an embodiment of the invention discussed in Example 1.
  • Figure 6 shows the conversion result of electropherogram into character coding in accordance with an embodiment of the invention.
  • Figure 7 shows a comparison result of using character coding and pair- wise alignment in accordance with an embodiment of the invention.
  • Figure 8 shows an electropherogram in accordance with an embodiment of the invention discussed in Example 2.
  • Figure 9 shows an electropherogram in accordance with an embodiment of the invention discussed in Example 3.
  • Figure 10 shows electropherograms in accordance with an embodiment of the invention discussed in Example 4.
  • Embodiments of the invention include a method of profiling nucleotide sequences from a sample to identify the sample based on its genomic information without performing full DNA sequencing. Rather than full DNA sequencing, a profile may be generated based on the relative position of the nucleotide bases. The positions of one, two, or three of the four nucleotides are used to generate the profile, but not all four. If all four were used, then full DNA sequencing would have been performed. The profiling pattern of differentiating genetic elements can thereby be used to identify a sample of interest, without performing full DNA sequencing.
  • the nucleotide sequence can be of any base length sufficient to provide a unique signature to the sample, and can include nucleic acids.
  • a sample such as a microorganism (e.g., bacteria, fungi, protozoans, algae, viruses) or a gene
  • a microorganism e.g., bacteria, fungi, protozoans, algae, viruses
  • Embodiments of the invention are useful to differentiate pathogenic microorganisms, aid in the selection of antimicrobial agents, and, in turn, decrease morbidity and mortality because of the application of more prompt and targeted therapy.
  • Microorganism DNA can be extracted from patient samples such as blood, tissue, urine, etc. and used to determine the identification of the infecting microorganism with embodiments of the invention.
  • the accurate identification of certain genes and genetic mutations is also an important facture influencing therapy selection.
  • mutation in BRCAl and BRC A2 genes significantly increase a person's risk of developing breast cancer.
  • a mutation in a gene corresponds to a change in the DNA sequence, such as nucleotide(s) insertion, deletion, exchange, and/or base order change.
  • Embodiments of the invention are useful for identifying these changes without performing full DNA sequencing of the sample.
  • the gene can be amplified and cleaved, and a genetic profile is obtained.
  • the genetic disorder can be identified by comparing the genetic profile to a reference library.
  • cleavage reactions targeted to one of the four nucleotides will generate enough data to identify medically-important samples.
  • cleavage reactions targeted at more than one nucleotide, but less than four can be used to create the signature.
  • a size-based separation and detection can then be performed. The pattern generated from the separation and detection can be used to identify the sample without going through base calling to identify the full DNA sequence.
  • Embodiments of the invention are useful for identifying any clinically relevant samples, such as microorganisms or other genetic organisms that provide nucleotide sequences, which in turn, provide nucleic acids, genes and other sequences.
  • Gram positive bacteria Gram negative bacteria
  • fungi fungi
  • yeasts yeasts
  • molds and viruses
  • any form such as cocci, rods, and spirochetes.
  • microorganisms known or suspected of causing disease such as enteric pathogens.
  • microorganisms include the genus Corynebacterium, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, alymmatobacte ⁇ um, Brucella, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia, Mycoplasma, Streptococcus (e.g., Group A, B, C, D or G Streptococcus, S.
  • pneumoniae S. pyogenes, S. agalactiae, S. faecalis, S. faecium, S. durans
  • Neisseria e.g., JV. gonorrheae, N. meningitidis
  • Staphylococcus e.g., S. aureus, S. epidermidis
  • Gardnerella Mycobacterium (e.g., M. tuberculosis, M. bovis, M. ulcerans, M. leprae), Listeria, Bordetella (e.g., B. pertusis, B. parapertusis, B.
  • bronchiseptica Escherichia, Shigella, Haemophilus (e.g., H. influenzae, H. aegyptius, H. parainfluenzae, H. ducreyi), Salmonella, Citrobacter, Proteus (e.g., P. mirabilis, P. vulgaris, Yersinia, Kleibsiella, Serratia (e.g., S. marcessens, S. liquefaciens), Vibrio cholera, Shigella (e.g., S. dysenterii, S.
  • Haemophilus e.g., H. influenzae, H. aegyptius, H. parainfluenzae, H. ducreyi
  • Salmonella Citrobacter
  • Proteus e.g., P. mirabilis, P. vulgaris, Yersinia, Kleibsiella, Serratia (e.g., S. marcessen
  • DNA templates from samples of interest undergo PCR amplification before chemical cleavage.
  • PCR amplification before chemical cleavage.
  • only one of the two primers is labeled with a fluorescent dye.
  • the resulting PCR product is labeled at one end of one of the DNA strands only. Any fragments after the chemical cleavage that still contain this fluorescent label can be detected with size-based separation (e.g., electrophoresis separation) and fluorescence detection.
  • size-based separation e.g., electrophoresis separation
  • fluorescence detection for example, consider a section of DNA from Staphylococcus aureus that has a sequence:
  • * represents the fluorescent dye label
  • the number in parentheses at the end of each sequence indicates the cleaved nucleotide base position.
  • the fragment generated is detected based on the 5' fluorescent label (*).
  • the respective 3' fragment generated by cleavage would not be detected, since it does not contain a fluorescent label:
  • additional base cleavage can be performed if cleavage at a single base does not provide sufficient information to definitively identify the sample.
  • Some embodiments of the invention provide for nucleotide sequence profiling for sample identification, obtained by a method comprising one or more of the following steps:
  • a nucleotide sequence (e.g., DNA) template from a sample of interest can be obtained by any suitable method.
  • the target is a microorganism presented in a culture (e.g., blood culture)
  • sufficient nucleotide sequences may be obtained by performing a simple lysis reaction. This may be done through mechanical cellular disruption (e.g., bead and/or ultrasonication), chemical methods (e.g., sodium hydroxide lysis), thermal disruption, or a combination of these methods.
  • a microorganism is sought directly from a clinical specimen (i.e., a complex matrix) wherein limited copy numbers are present, then a nucleotide sequence extraction procedure may be necessary.
  • a solid phase extraction method can be used in which the DNA binds to a solid phase bead or column depending on the pH and buffer used. The bead or column can then be washed to remove any impurities. An elution buffer can be used to remove the DNA from the solid phase surface and the eluted DNA can be collected for future analysis.
  • the template can be amplified using any suitable method.
  • PCR can be performed on the template using broad-range primers that are designed to amplify a genomic region that contains sufficient genetic variability to afford sample identification.
  • the broad range primers may be the so-called "universal" primers, which will amplify any sample, such as a microorganism, in a large group (e.g., pan- fungal primers) or they may be for a select subset of microorganisms (e.g., the staphylococci). Regardless of the design, these primers function in the PCR to amplify a particular section of genomic nucleotide sequence (e.g., DNA) in which there is species-specific information.
  • genomic nucleotide sequence e.g., DNA
  • primers in the pair is labeled with a dye for later DNA cleavage fragment identification; or both primers are labeled with different dyes for later DNA cleavage fragment identification.
  • primers without dye labeled could be used to perform PCR and a derivative reaction could be performed on the PCR product to label one or both DNA chain(s).
  • labeled nucleotides could be used.
  • step b) Performing chemical cleavage on the products obtained in step b), in such a way that only one of the four nucleotides is cut.
  • Chemicals can be used to selectively modify and cleave the nucleotide sequence strands (e.g., DNA) at A, G, T, or C. Use of limiting concentrations of such chemicals allows partial digestion of the DNA at different base positions.
  • step d) Or, alternatively, performing chemical cleavage on the products obtained in step b) at multiple nucleotide positions.
  • chemicals can be used to selectively modify and cleave the DNA strands at more than 1, but less than 4, (i.e., two or three) nucleotide positions.
  • Size-based separation can be used to separate the product. Any useful method can be used.
  • electrophoresis can be used to separate the digested nucleotide sequence fragments based on their sizes. Examples include slab gel electrophoresis, capillary gel electrophoresis, multiplexed capillary gel electrophoresis, and microchip based gel electrophoresis.
  • electrophoresis instruments such as the DNA PROFiler or cePRO 9600 Fl, available from Advanced Analytical Technologies, Inc., Ames, IA, assignee of the present application, can be used for the size-based separation step.
  • the cleavage products can be detected by a fluorescent signal in embodiments where digested nucleotide sequence fragments contain a dye label.
  • Many dyes can be used to label the primers, such as any dye useful for nucleotide sequencing, such as FAM, JOE, TAMRA, ROX, and energy transfer dyes.
  • a profile such as the profile shown in Figure 5
  • the electropherograms can be generated.
  • step h) Comparing the profile generated in step g) to the data base for sample identification.
  • the created profile can be compared to a data base to identify the sample.
  • a comparison algorithm can be used to compare the sample profile to a database for sample identification.
  • the profile is compared to a known database to identify the sample.
  • Any pattern recognition algorithm useful for comparing the nucleotide sequence profile to the database can be used. For example, the migration time of each peak identified in the electropherogram can be used to compare the pattern with the use of principle components analysis. See Duarte et al. "Application of Chemometrics in Separation Science", J. Liq. Chromatogr. & Related Tech., 2006, Vol. 29, pp. 1143-1176, the relevant contents of which are hereby incorporated by reference.
  • dynamic programming to align sample electropherogram to a standard electropherogram may be used. See Guillo et al. "Micellar Electrokinetic Capillary Chromatography And Data Alignment Analysis: A New Tool In Urine Profiling", J. Chromatogr. A, 2004, Vol. 1027, pp. 203-212, the relevant contents of which are hereby incorporated by reference.
  • Such an approach uses dynamic programming to correct the migration time shifts and to provide a similarity score between whole electropherograms.
  • Another approach uses the peak height information in the coded sequences to improve the reliability of the searching algorithm. See Ceballos et al.
  • the electropherogram may be smoothed first to reduce noise. Algorithms such as moving average, wavelet, FFT, or Savitzky-Golay filters can be used to smooth out the data to reduce noise. 2.
  • the peaks of the electropherogram are identified, as shown in Figure 6 with vertical lines. First and second derivatives can be used to identify each peak position. 3. The peaks are divided into groups depending on their intensities. For example,
  • Figure 6 marks the larger intensity peaks as G while smaller peaks as C. 4.
  • the spacing between adjacent identified peaks is calculated.
  • a distribution or histogram from the spacing values can be created.
  • a gap threshold can then be estimated to determine the standard peak spacing between adjacent peaks.
  • the adjacent peak spacing is, for example, two times larger than the threshold, a gap can be identified and marked as T, as shown in Figure 6.
  • the adjacent peaks spacing is, for example, three times larger than the threshold, then the gap can be identified and marked with two Ts, and so on.
  • electropherograms from a reference database and an unknown sample are transformed from an analogue signal to character coding.
  • the electropherogram in Figure 6 transformed into character coding by using the previous described method produces the following result:
  • G represents the peaks larger than a threshold while C represents the peaks smaller than the threshold and T represents a gap.
  • a pair- wise alignment algorithm such as Needleman-Wunsch global alignment or Smith- Waterman local alignment, can then be used to compare the character codings from the sample electropherogram to the database reference electropherogram. Such a method can be used to determine the difference between the electropherograms, as shown in Figure 7.
  • embodiments of invention use the pattern generated from cleaved nucleotide sequence fragments to form a fingerprint for sample identification. With such a method, there is no need to identify individual fragments for nucleotide base calling as in full DNA sequencing.
  • Figure 5 shows the overlay of the two electropherograms.
  • This reaction mainly cleaves at thymine (T) base positions.
  • T thymine
  • the two species may be differentiated and identified by comparing their partial DNA profiling without knowing the full DNA profile for the section of genome.
  • Example 2 Generating a DNA profile through a chemical cleavage method on multiple base positions.
  • DNA templates from Candida species C. parapsilosis, C. glabrata, C. albicans, and C. tropicalis were used for PCR with a universal primer as described in Example 1.
  • the DNA were modified and digested with N-dimethylformamide and 3 niM MnCl 2 at 110°C for 30 minutes.
  • the same method described in Example 1 for sample purification was used before sample injection and electrophoresis separation.
  • Figure 8 shows the electropherograms for each of the samples. For a 5 '-end-labeled DNA fragment, each random degradation creates two fragments that contained the labeled dye. See Negri et a "One-Step, One-Lane Chemical DNA Sequencing by N-
  • ITS1/ITS4 was labeled with the fluorescent dye fluorescein.
  • ITS 1 is a forward primer having the following sequence: TCCGT AGGTGAACCTGCGG.
  • ITS4 is a reverse primer having the following sequence: GCATATCAATAAGCGGAGGA.
  • Figure 9 shows the electropherograms for each of the cleavage samples. This reaction preferably cleaves DNA at the G and A positions with moderate cleavage at the C position and insignificant cleavage at the T position. From Figure 9, one can identify the difference between these species.
  • the forward primer ISTl could be labeled with FAM (carboxyfluorescein), which has a maximum emission at -520 nm and the reverse primer IST4 labeled with Cy-5, which has a maximum emission at -670 nm.
  • FAM carboxyfluorescein
  • Cy-5 which has a maximum emission at -670 nm.
  • the fluorescent signals generated would then come from both dye labels. If the fluorescence detection system is capable of identifying the different emission wavelength, then the two signals could separate from one another.
  • the detection system examines two wavelength bands, one centrals at 520 nm with bandwidth ⁇ 40 nm and the other centrals at 670 nm ⁇ 40 nm, then one could determine which fragments came from which strand of DNA. Then, the electropherograms could be re-constructed according to the emission wavelength, i.e., one electropherogram shows the fragments having emission at 520 nm and the other electropherogram shows the fragments having emission at 670 nm. Since both strands of DNA are complementary to each other, in this manner one could get twice the amount of information, which could be used to identify the microorganism, in contrast to generating a DNA profile from one end of the DNA alone.
  • the following DNA sequence can be selected as primers for the PCR amplification: 5 ' -TATTCTC AATC ACTGGTCGT-3 ' 5'-AGCTTCAGCGTAGTCTA-S'.
  • 5'-TATTCTCAATCACTGGTCGT-S' can be labeled with a fluorescence dye such as FAM in the 5' position and 5'-AGCTTCAGCGTAGTCTA- 3' can be labeled with a fluorescence dye such as Cy-5 at the 5' position.
  • a fluorescence dye such as FAM
  • 5'-AGCTTCAGCGTAGTCTA- 3' can be labeled with a fluorescence dye such as Cy-5 at the 5' position.
  • a capillary electrophoresis with fluorescence detection system can be used to separate and differentiate the fragments that labeled with different dyes simultaneously.
  • Figure 10 shows the separation results.
  • the bottom trace represents the blue emission wavelength from 480 nm to 560 nm and the top trace represents the red emission wavelength from 620 nm and up.
  • the 480 nm to 560 nm represents the emission from FAM labeled DNA fragments while the other wavelength represents the emission from Cy-5 labeled DNA fragments.
  • the traces have different patterns since each trace represents each DNA strand at the opposite direction. This two-color method allows better coverage of the PCR amplicons to reveal the more subtle change of the DNA sequences in between sub-species. If the fragments from one strand could not differentiate the microorganism, the fragments from the other strand may provide additional information for microorganism identification.
  • DNA molecules produced from the PCR are chemically cleaved at a specific location(s).
  • a DNA profile is generated that is based on the DNA sequence.
  • the DNA profile can be used to identify the microorganism species from a database.
  • a "most likely match" or percent identity value could still be generated to indicate the possible species for the microorganism.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de profilage d'un échantillon pour identification. Le procédé comprend les étapes consistant à réaliser moins de quatre réactions de clivage chimique spécifiques à des nucléotides pour obtenir des fragments de séquence nucléotidique, réaliser une séparation de taille des fragments, détecter la séparation des fragments, générer un profil fondé sur la détection et comparer le profil à une base de données pour identifier l'échantillon.
PCT/US2008/087434 2007-12-18 2008-12-18 Système et procédé pour le profilage de séquences nucléotidiques pour l’identification d’échantillons WO2009117031A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1460907P 2007-12-18 2007-12-18
US61/014,609 2007-12-18

Publications (2)

Publication Number Publication Date
WO2009117031A2 true WO2009117031A2 (fr) 2009-09-24
WO2009117031A3 WO2009117031A3 (fr) 2009-12-17

Family

ID=41091435

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/087434 WO2009117031A2 (fr) 2007-12-18 2008-12-18 Système et procédé pour le profilage de séquences nucléotidiques pour l’identification d’échantillons

Country Status (2)

Country Link
US (1) US8101353B2 (fr)
WO (1) WO2009117031A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021206539A1 (fr) * 2020-04-11 2021-10-14 Universiti Malaya Dispositif de profilage et d'identification d'un oligonucléotide

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103782157B (zh) * 2011-08-30 2016-01-20 奥林巴斯株式会社 包含胰液的生物试样中的目标粒子的检测方法
JP6775014B2 (ja) 2015-11-06 2020-10-28 キアゲン サイエンシーズ， エルエルシー ヌクレオチド類似体
AU2017237199B2 (en) 2016-03-25 2020-11-05 Karius, Inc. Synthetic nucleic acid spike-ins
JP7091325B2 (ja) * 2016-11-04 2022-06-27 キアゲン サイエンシーズ， エルエルシー チオール含有切断試薬および酸化的洗浄液
WO2020106987A1 (fr) * 2018-11-21 2020-05-28 Karius, Inc. Détection et prédiction de maladie infectieuse
CA3118990A1 (fr) 2018-11-21 2020-05-28 Karius, Inc. Procedes, systemes et compositions de bibliotheque directe

Family Cites Families (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5064754A (en) * 1984-12-14 1991-11-12 Mills Randell L Genomic sequencing method
US4865968A (en) * 1985-04-01 1989-09-12 The Salk Institute For Biological Studies DNA sequencing
US5683874A (en) * 1991-03-27 1997-11-04 Research Corporation Technologies, Inc. Single-stranded circular oligonucleotides capable of forming a triplex with a target sequence
US5308751A (en) * 1992-03-23 1994-05-03 General Atomics Method for sequencing double-stranded DNA
DE4218152A1 (de) * 1992-06-02 1993-12-09 Boehringer Mannheim Gmbh Simultane Sequenzierung von Nukleinsäuren
US5503980A (en) * 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization
US5888780A (en) * 1992-12-07 1999-03-30 Third Wave Technologies, Inc. Rapid detection and identification of nucleic acid variants
WO1995010771A1 (fr) * 1993-10-14 1995-04-20 Bio-Rad Laboratories, Inc. Suppression de l'endosmose electrique pendant une electrophorese en milieu polymere depourvu de gel a l'aide de polymeres charges
US5512458A (en) * 1994-02-25 1996-04-30 W. R. Grace & Co.-Conn. Method of using mobile priming sites for DNA sequencing
US5552278A (en) * 1994-04-04 1996-09-03 Spectragen, Inc. DNA sequencing by stepwise ligation and cleavage
DK0843735T4 (da) * 1995-07-11 2007-01-22 Univ College Cork Nat Univ Ie Glycolasemedieret påvisning af kendte nukleotidsekvenser
CA2185590A1 (fr) * 1995-09-18 1997-03-19 Kazunori Okano Methode de sequencage d'adn et methode de preparation d'un echantillon d'adn
US5858671A (en) * 1996-11-01 1999-01-12 The University Of Iowa Research Foundation Iterative and regenerative DNA sequencing method
US6117634A (en) * 1997-03-05 2000-09-12 The Reagents Of The University Of Michigan Nucleic acid sequencing and mapping
JP2001512700A (ja) * 1997-08-07 2001-08-28 キュラジェン コーポレイション 公知の末端配列に厳密にハイブリダイズするサブ配列および未同定の配列にハイブリダイズするサブ配列を含むオリゴヌクレオチドの使用による核酸配列の検出および確認
US6087099A (en) * 1997-09-08 2000-07-11 Myriad Genetics, Inc. Method for sequencing both strands of a double stranded DNA in a single sequencing reaction
JP4317953B2 (ja) * 1998-01-22 2009-08-19 独立行政法人理化学研究所 Ｄｎａの塩基配列決定方法
US7270958B2 (en) * 1998-09-10 2007-09-18 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
US6440705B1 (en) * 1998-10-01 2002-08-27 Vincent P. Stanton, Jr. Method for analyzing polynucleotides
WO2001023610A2 (fr) * 1999-09-29 2001-04-05 Solexa Ltd. Sequençage de polynucleotides
EP2100971A3 (fr) * 2000-07-07 2009-11-25 Visigen Biotechnologies, Inc. Détermination de séquence en temps réel
AU7814101A (en) * 2000-08-24 2002-03-04 Bio Rad Laboratories Dry powder formulation for protein labeling
AU2003244370A1 (en) * 2002-02-05 2003-09-02 Baylor College Of Medecine Substituted 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene compounds for 8-color dna sequencing
EP1487993A2 (fr) * 2002-02-11 2004-12-22 Genome Express Analyse discriminative d'acides nucleiques en utilisant des signatures de sequence de clones
EP1369493B1 (fr) * 2002-06-05 2006-09-13 Epigenomics AG Procédé pour la détermination quantitative du degré de methylation de cytosines en positions CpG
US20050115837A1 (en) * 2003-12-01 2005-06-02 Dean Burgi Analyte identification in transformed electropherograms

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021206539A1 (fr) * 2020-04-11 2021-10-14 Universiti Malaya Dispositif de profilage et d'identification d'un oligonucléotide

Also Published As

Publication number Publication date
US8101353B2 (en) 2012-01-24
WO2009117031A3 (fr) 2009-12-17
US20100209908A1 (en) 2010-08-19

Similar Documents

Publication Publication Date Title
JP7586531B2 (ja) バイオマーカーおよびその使用
US8101353B2 (en) System and method for nucleotide sequence profiling for sample identification
US5753467A (en) Method for the identification of microorganisms by the utilization of directed and arbitrary DNA amplification
US6972174B2 (en) Method for detecting single nucleotide polymorphisms (SNP's) and point mutations
US6150105A (en) Methods of screening nucleic acids for nucleotide variations
KR101038137B1 (ko) 서열 차이를 감지하는 방법
US7189512B2 (en) Methods for variation detection
EP1105537B1 (fr) Procede relatif a la determination des variations de sequence polynucleotidique
Käller et al. Arrayed identification of DNA signatures
US20080172183A1 (en) Systems and methods for methylation prediction
JP2005530508A (ja) プライマー伸長反応および多型検出反応をモニターするための方法および組成物
CN117778568A (zh) 鉴别胃癌的标志物及应用
US7585626B1 (en) Methods for nucleic acid amplification
US20120053064A1 (en) Determining the identity of terminal nucleotides
Gut An overview of genotyping and single nucleotide polymorphisms (SNP)
CN109312397A (zh) Penta E基因座多态性人体鉴定
van Pelt-Verkuil et al. Variants and Adaptations of the Standard PCR Protocol
US20040121394A1 (en) Method for determining polynucleotide sequence variations
WO2009143590A2 (fr) Protocol de détection de séquence d'insertion

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08873484

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08873484

Country of ref document: EP

Kind code of ref document: A2

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载